Brucella melitensis is a human and animal bacterial pathogen of global significance. Although bacterial factors mediating the host-pathogen interaction have been revealed, host factors that are important for B. melitensis entry and replication in mammalian cells remain obscure. We have recently exploited a novel Drosophila melanogaster S2 cell model of Brucella infection to perform a pilot screen for host factors that support the replication of this intracellular pathogen. Our screen uncovered genes that have already been established as being important for B. melitensis strain 16M (Bm16M) entry and intracellular trafficking in mammalian cells. In addition, our screen uncovered several novel hits that were validated in mammalian cell models. Here, we propose to exploit these findings to perform a large-scale RNAi screen for additional host factors. Specifically, we aim: (1) To perform a large-scale screen for RNAis that disrupt or enhance B. melitensis infection of Drosophila S2 cells;(2) To classify hits into phenotypic and functional categories, and to compare the results obtained to those from the recently completed Mycobacterium fortuitum and Listeria monocytogenes screens. Finally, we shall employ siRNA technology to examine whether the mammalian orthologs of the hits obtained in our Drosophila S2 cell screen mediate Bm16M infection of mammalian cells. Taken together, these experiments will define additional host factors that support the uptake and replication of B. melitensis into animal cells, and thereby provide new insights into the molecular mechanisms mediating this important host-pathogen interaction. This proposal shall identify and characterize novel host factors mediating Brucella melitensis infection of animal cells. We expect that this effort shall therefore benefit public health by contributing significantly to our understanding of this pathogen of global significance, and by discovering potential protein targets for possible therapeutic intervention. Public Health Relevance. This proposal shall identify and characterize novel host factors mediating Brucella melitensis infection of animal cells. We expect that this effort shall therefore benefit public health by contributing significantly to our understanding of this pathogen of global significance, and by discovering potential protein targets for possible therapeutic intervention.